1. Field of the Invention
This invention relates to a method of assaying physiologically active substances. More particularly, it relates to a method of assaying physiologically active substances involved in the process of plasma kallikrein formation.
2. Description of the Prior Art
Kallikrein is a group of protease widely distributed in the plasma and tissues of animals, and is known to participitate in an enzyme reaction system called kallikrein-kinin system.
This kallikrein-kinin system is closely related to many other enzyme reaction systems, such as the renin-angiotensin system, the blood coagulation system, the fibrinolysis system, the complement system, and the arachidonate cascade producing prostaglandin, leukotriene and thromboxane, as well as to the behavior of catecholamine, thus playing an important role for function control in living bodies. To be more specific, it is deeply concerned, through other enzyme reaction systems, with blood pressure control, control actions relating to blood coagulation fibrinolysis-complement system, and peripheral blood circulation improvement and other control actions exerted by various physiologically active substances produced from the arachidonate cascade.
It is known that plasma kinin, which is a product of the kallikrein-kinin system, shows various physiological actions, such as hypotensive action as a result of vasodilation, enhancement of vascular permeability, contraction and relaxation of smooth muscles, development of pains, leucocytes chemotaxis, and liberation of catecholamine from the adrenal cortex. It is also known that plasma kinin acts as a mediator for acute inflammations, including allergic reactions. Thus the presence of plasma kinin in living bodies is of great significance.
Hence, establishing a simple method for correctly evaluating the action of substances involved in the formation of kallikrein ( substances that retard or accelerate the formation of kallikrein ) would be of great use in learning the aforementioned actions for function control in living bodies and also in developing new drugs having such actions.
The kallikrein-kinin system involves a series of enzyme reactions as described below.
Blood coagulation factor XII ( Hageman factor; hereinafter abbreviated as F-XII ) is the substance which plays the major role in this enzyme reaction system. F-XII in the plasma is activated upon contact with a negatively charged substance ( such as glass, kaolin and elaidic acid ) or with a substance existing in living bodies ( such as collagen, homocystine, platelet membrane and sulfated glycolipid ), or as a result of nociceptive stimulation upon tissues. The resulting activated F-XII ( F-XIIa ) then acts upon prekallikrein present in the plasma to convert it into kallikrein, which in turn acts upon high-molecular-weight kininogen in the plasma, liberating bradykinin which is a nonapeptide.
Eventually, the kinin thus formed causes inflammations and pains, and exerts various influences on living bodies through action upon the arachidonate cascade.
In-vitro simulation of the aforementioned series of enzyme reactions related to the kallikrein-kinin system has led us to find that the reaction system adopted in this invention provides a simple, highly reliable, and most effective method for assaying physiologically active substances. This invention was accomplished based on these findings.